ABSTRACT The growth and proliferation rates of cells are proportional to the rate of ribosome biosynthesis. This relationship is emphasized in cancer, where the continuous demand for ribosome synthesis is prevalent. The first step in ribosome biosynthesis is the transcription of the ribosomal (r) DNA by RNA polymerase I (Pol I); in cancers, this process is deregulated. Despite these established connections between ribosome biosynthesis and cancer cell proliferation, the regulatory networks that control transcription elongation at the rDNA locus remain poorly defined. We have discovered a new regulatory checkpoint that monitors transcription perturbations at the rDNA and is resolved by the degradation of the Pol I catalytic subunit RPA194. This checkpoint is activated by our newly identified chemical inhibitor of Pol I (BMH-21). The goal of this application is to characterize this checkpoint by defining factors and molecular mechanism(s) by which it is activated. Furthermore, this work will identify the molecular basis for the degradation of the enzyme, and the reciprocal regulation of the Pols I and II at the rDNA locus. This proposal will pursue three primary goals: (1) Characterize factors that monitor Pol I elongation; (2) Define how the Pol I elongation checkpoint is activated and enforced and how it impacts Pol II; and (3) Identify ubiquitin-proteasome system components that mediate the degradation of RPA194. To achieve these goals, we will pursue three specific aims. In these aims, BMH-21 and other epigenetic drugs will be used as tools to study the polymerase-selective checkpoint. Aim 1 characterizes factors identified in yeast and mammalian genome-wide screens and specifies how they regulate Pol I transcription. Aim 2 implements biochemically defined transcription assays and genomic analyses to identify how Pol I transcription perturbations activate the checkpoint, and its impact on polymerase selectivity. Aim 3 defines the roles of ubiquitin-proteasome system factors in controlling the stability and transcription of Pol I. In all, these studies provide unprecedented insight into the regulation of Pol I transcription and the reorganization of nuclear transcription upon inactivation of Pol I. An in-depth, mechanistic understanding of Pol I transcription regulation will support strategies seeking therapeutic control of deregulated rRNA synthesis in human disease.